Bottom Line:
Electrically triggered action potentials in the giant alga Chara corallina are associated with a transient rise in the concentration of free Ca(2)+ in the cytoplasm (Ca(2)+(cyt)).The present measurements of Ca(2)+(cyt) during membrane excitation show that stimulating pulses of low magnitude (subthreshold pulse) had no perceivable effect on Ca(2)+(cyt).Assuming that inositol-1,4,5,-trisphosphate (IP(3)) is the second messenger in question, the present data provide the major rate constants for IP(3) metabolism.

ABSTRACTElectrically triggered action potentials in the giant alga Chara corallina are associated with a transient rise in the concentration of free Ca(2)+ in the cytoplasm (Ca(2)+(cyt)). The present measurements of Ca(2)+(cyt) during membrane excitation show that stimulating pulses of low magnitude (subthreshold pulse) had no perceivable effect on Ca(2)+(cyt). When the strength of a pulse exceeded a narrow threshold (suprathreshold pulse) it evoked the full extent of the Ca(2)+(cyt) elevation. This suggests an all-or-none mechanism for Ca(2)+ mobilization. A transient calcium rise could also be induced by one subthreshold pulse if it was after another subthreshold pulse of the same kind after a suitable interval, i.e., not closer than a few 100 ms and not longer than a few seconds. This dependency of Ca(2)+ mobilization on single and double pulses can be simulated by a model in which a second messenger is produced in a voltage-dependent manner. This second messenger liberates Ca(2)+ from internal stores in an all-or-none manner once a critical concentration (threshold) of the second messenger is exceeded in the cytoplasm. The positive effect of a single suprathreshold pulse and two optimally spaced subthreshold pulses on Ca(2)+ mobilization can be explained on the basis of relative velocity for second messenger production and decomposition as well as the availability of the precursor for the second messenger production. Assuming that inositol-1,4,5,-trisphosphate (IP(3)) is the second messenger in question, the present data provide the major rate constants for IP(3) metabolism.

Figure 6: Strength-duration relationship for electrical stimulation of transient calcium rise. One cell was stimulated with two different protocols. Stimulation was either performed with a single pulse (circles) or as in Fig. 4 with double subthreshold pulses (squares). In the latter case, only the trailing pulse was varied in duration and strength while the parameters of the leading pulse (I = 2 μA, t = 200 ms) as well as Δt2 (500 ms) were kept constant. Shown are strength-duration values that were effective (closed symbols) or ineffective (open symbols) for inducing a transient calcium rise. In the case of double pulse stimulation, only the strength-duration values of the trailing pulse are considered in the plot. Fitting the data with a hyperbolic function yielded a minimum current I0 = 1.2 μA and a minimum charge qmin = 135 nC for single pulse stimulation. For the trailing pulse in the double pulse protocol, the fit yielded I0 = 1.3 μA and qmin = 114 nC.

Mentions:
To further test the hypothesis that two subthreshold pulses could be additive in their ability to stimulate Ca2+ mobilization, we compared the minimum charge (qmin) required for stimulation with single and double pulses. Therefore, one cell was stimulated (as in Fig. 4) with a double pulse protocol. However, in this case, strength and duration of the second pulse were varied, whereas the parameters of the first pulse as well as Δt2 were kept constant. For comparison, the same cell was also stimulated with single pulses of variable strength and duration. The plot in Fig. 6 illustrates the strength-duration relationship in one cell for the two different modes of stimulation, i.e., stimulation with a single pulse (closed symbols) and stimulation with a variable second pulse after a leading constant pulse (open symbols). Fitting of both data sets with yielded very similar values for I0. However, the qmin value from the double pulse stimulation was 1.19 times lower than that for single pulse stimulation. The same result was confirmed in three similar experiments showing that the qmin required for effective stimulation was on average 1.2 ± 0.06 times smaller, when the stimulating pulse was preceded by a subthreshold pulse. These data and the finding that the strength-duration plot for the second pulse shows the same hyperbolic relationship as that obtained for single pulse stimulation is best explained by the fact that individual pulses are indeed additive.

Figure 6: Strength-duration relationship for electrical stimulation of transient calcium rise. One cell was stimulated with two different protocols. Stimulation was either performed with a single pulse (circles) or as in Fig. 4 with double subthreshold pulses (squares). In the latter case, only the trailing pulse was varied in duration and strength while the parameters of the leading pulse (I = 2 μA, t = 200 ms) as well as Δt2 (500 ms) were kept constant. Shown are strength-duration values that were effective (closed symbols) or ineffective (open symbols) for inducing a transient calcium rise. In the case of double pulse stimulation, only the strength-duration values of the trailing pulse are considered in the plot. Fitting the data with a hyperbolic function yielded a minimum current I0 = 1.2 μA and a minimum charge qmin = 135 nC for single pulse stimulation. For the trailing pulse in the double pulse protocol, the fit yielded I0 = 1.3 μA and qmin = 114 nC.

Mentions:
To further test the hypothesis that two subthreshold pulses could be additive in their ability to stimulate Ca2+ mobilization, we compared the minimum charge (qmin) required for stimulation with single and double pulses. Therefore, one cell was stimulated (as in Fig. 4) with a double pulse protocol. However, in this case, strength and duration of the second pulse were varied, whereas the parameters of the first pulse as well as Δt2 were kept constant. For comparison, the same cell was also stimulated with single pulses of variable strength and duration. The plot in Fig. 6 illustrates the strength-duration relationship in one cell for the two different modes of stimulation, i.e., stimulation with a single pulse (closed symbols) and stimulation with a variable second pulse after a leading constant pulse (open symbols). Fitting of both data sets with yielded very similar values for I0. However, the qmin value from the double pulse stimulation was 1.19 times lower than that for single pulse stimulation. The same result was confirmed in three similar experiments showing that the qmin required for effective stimulation was on average 1.2 ± 0.06 times smaller, when the stimulating pulse was preceded by a subthreshold pulse. These data and the finding that the strength-duration plot for the second pulse shows the same hyperbolic relationship as that obtained for single pulse stimulation is best explained by the fact that individual pulses are indeed additive.

Bottom Line:
Electrically triggered action potentials in the giant alga Chara corallina are associated with a transient rise in the concentration of free Ca(2)+ in the cytoplasm (Ca(2)+(cyt)).The present measurements of Ca(2)+(cyt) during membrane excitation show that stimulating pulses of low magnitude (subthreshold pulse) had no perceivable effect on Ca(2)+(cyt).Assuming that inositol-1,4,5,-trisphosphate (IP(3)) is the second messenger in question, the present data provide the major rate constants for IP(3) metabolism.

ABSTRACTElectrically triggered action potentials in the giant alga Chara corallina are associated with a transient rise in the concentration of free Ca(2)+ in the cytoplasm (Ca(2)+(cyt)). The present measurements of Ca(2)+(cyt) during membrane excitation show that stimulating pulses of low magnitude (subthreshold pulse) had no perceivable effect on Ca(2)+(cyt). When the strength of a pulse exceeded a narrow threshold (suprathreshold pulse) it evoked the full extent of the Ca(2)+(cyt) elevation. This suggests an all-or-none mechanism for Ca(2)+ mobilization. A transient calcium rise could also be induced by one subthreshold pulse if it was after another subthreshold pulse of the same kind after a suitable interval, i.e., not closer than a few 100 ms and not longer than a few seconds. This dependency of Ca(2)+ mobilization on single and double pulses can be simulated by a model in which a second messenger is produced in a voltage-dependent manner. This second messenger liberates Ca(2)+ from internal stores in an all-or-none manner once a critical concentration (threshold) of the second messenger is exceeded in the cytoplasm. The positive effect of a single suprathreshold pulse and two optimally spaced subthreshold pulses on Ca(2)+ mobilization can be explained on the basis of relative velocity for second messenger production and decomposition as well as the availability of the precursor for the second messenger production. Assuming that inositol-1,4,5,-trisphosphate (IP(3)) is the second messenger in question, the present data provide the major rate constants for IP(3) metabolism.